Imatinib immunoassay

ABSTRACT

Novel conjugates and immunogens derived from imatinib and monoclonal antibodies generated by these immunogens are useful in immunoassays for the quantification and monitoring of imatinib or its pharmacologically active salts in biological fluids.

FIELD OF THE INVENTION

This invention relates to the field of immunoassays for determining thepresence and/or quantifying the amount of imatinib or itspharmacologically active salts in human biological fluids in order torapidly determine optimal drug concentrations during chemotherapy.

BACKGROUND OF THE INVENTION

Cancer is a term used to describe a group of malignancies that all sharethe common trait of developing when cells in a part of the body begin togrow out of control. Most cancers form as tumors, but can also manifestin the blood and circulate through other tissues where they grow. Cancermalignancies are most commonly treated with a combination of surgery,chemotherapy, and/or radiation therapy. The type of treatment used totreat a specific cancer depends upon several factors including the typeof cancer malignancy and the stage during which it was diagnosed.

Imatinib has the following formula:

and its salts, particularly imatinib mesylate, are one of the morecommonly used chemotherapeutic agents for treatment of Philadelphiachromosome positive chronic myeloid leukemia in blast phase, acceleratedphase or chronic phase. (Gleevec package insert, NovartisPharmaceuticals Corporation, July 2004).

Imatinib has been shown to have up to a 16 fold inter-patientvariability in trough concentrations and that this variability canimpact efficacy. (Picard et. al. Blood 2007: 109; 3496-3499, Larson etal. Blood 2008, 111: 4022-4028, Demetri et. al. J Clin Oncol 2009, 27:3141-3147)

The preferred salt of imatinib is imatinib mesylate has the formula:

Since efficacy of imatinib is improved at higher trough levels and thatthe drug exhibits wide intra-patient pharmacokinetic variabilitymonitoring concentrations of this drug in blood and adjusting to targetlevels would be of value in increasing efficacy and minimizing toxicity.The degree of intra- and inter-individual pharmacokinetic variability ofimatinib and its salts has been reported to be 16 fold and is impactedby many factors, including:

-   -   Organ function    -   Genetic regulation    -   Disease state    -   Age    -   Drug-drug interaction    -   Time of drug ingestion    -   Compliance

As a result of this variability, equal doses of the same drug indifferent individuals can result in dramatically different clinicaloutcomes. The effectiveness of the same dosage of imatinib or its saltsvaries significantly based upon individual drug clearance and theultimate serum drug concentration in the patient. Therapeutic drugmanagement would provide the clinician with insight on patient variationin drug administration. With therapeutic drug management, drug dosagescould be individualized to the patient, and the chances of effectivelytreating the cancer without the unwanted side effects would be muchhigher.

In addition, therapeutic drug management of imatinib or its salts wouldserve as an excellent tool to ensure compliance (Henk, et al. Proc ASCO2006, abst. 6083, Feng, et al. Proc ASCO 2006, abst. 6038) inadministering chemotherapy with the actual prescribed dosage andachievement of the effective serum concentration levels. Routinetherapeutic drug management of imatinib or its salts would require theavailability of simple automated tests adaptable to general laboratoryequipment. The use of liquid chromatography (LC)-tandem massspectroscometry to determine the concentration of imatinib, imatinibsalts or their chemotherapeutic metabolites in human blood and plasmahas been described (Guetens, J Pharm Biomed Anal., 33(5):879-89 2003;Bakhtiar, J Chromatrography B, 768(2):325-340, 2002; Titier, Ther. Drug.Monit., (27)5:634-640, 2005). A LC method to determine the purity ofimatinib, imatinib salts or their chemotherapeutic metabolites(Vivekanand, J Pharm Biomed Anal., 28(6):1183-94, 2002) has also beendeveloped but was not used to determine levels in biological fluids.These methods are labor intensive, use expensive equipment and are notamenable to routine clinical laboratory use. An enzymatic assay formeasuring imatinib has been developed set forth in U.S. Pat. No.7,300,768. However there exists no simple immunoassay for determiningthe presence or quantifying the amount of imatinib in human biologicalfluids of patients treated with this chemotherapeutic agent.

As seen from the foregoing, there are no immunoassays for determiningthe presence and/or quantifying the amount of imatinib or itspharmacologically active salts in human biologically fluids. Routinetherapeutic drug management of imatinib and its pharmacologically activesalts by immunoassays would provide simple automated tests adapted tostandard laboratory equipment. However, in order to provide suchimmunoassays, antibodies specific to imatinib and its pharmacologicallyactive salts must be produced. The derivatives and immunogen used inthis assay must impart through these corresponding antibodies producedspecific reactivity to imatinib and its pharmacologically active saltswithout any substantial cross reactivity to therapeutically active orinactive or pharmacologically active or inactive metabolites of imatiniband their salts. In order to be effective in monitoring drug levels, theantibodies should be specific to imatinib and its pharmacologicallyactive salts and not cross reactive with N-desmethyl imatinib

The active metabolite of imatinib which occurs in samples of patientstreated with imatinib and its salts is N-desmethyl imatinib which hasthe formula:

It is this pharmacologically active metabolite which prevents accuratedetermination imatinib and its salts by immunoassays of samples ofpatients treated with imatinib and its salts. Therefore it is long beendesired to provide antibodies specific to imatinib and itspharmacologically active salts and not cross reactive with N-desmethylimatinib

SUMMARY OF INVENTION

In accordance with this invention, a new class of antibodies have beenproduced which are substantially selectively reactive to imatinib andits pharmacologically active salts so as to selectively bind to imatiniband its pharmacologically active salts without any substantial crossreactivity to the its pharmacologically active imatinib metabolite,N-desmethyl imatinib. By selectively reactivity, it is meant that thisantibody only reacts with the imatinib and its pharmacologically activesalts and does not substantially react with the pharmacologically activeimatinib metabolite, N-desmethyl imatinib The N-desmetyl imatinibmetabolite prevents an accurate determination by an immunoassay of thepresence and the amount of imatinib and its pharmacologically activesalts in human biological fluids.

It has been found that by using immunogens which are conjugates of acarrier containing an immunogenic polymer having a reactive thiol oramino functional group with a compound of the formula:

or its pharmacologically active salts.

-   -   wherein Y is an organic spacing group;        -   X is a terminal functional group capable of binding to said            carrier through said amino or thiol group and;        -   p is an integer from 0 to 1;

produce antibodies which are specific for imatinib or itspharmacologically active salts and do not substantially react with orbind to N-desmethyl imatinib. In addition these antibodies displaysubstantially no cross reactivity with any of its therapeutically activeor inactive imatinib metabolites

The provision of these antibodies which substantially selectively reactwith imatinib and its pharmacologically active salts and do not crossreact with N-desmethyl imatinib allows one to produce an immunoassaywhich can specifically detect and quantify so as to monitor imatinib andits pharmacologically active salts in the fluid samples of patientsbeing treated with imatinib or its pharmacologically active salts. Alsoincluded within this invention are reagents and kits for saidimmunoassay. The presence of the active metabolite of imatinib,N-desmethyl imatinib, is the major cause for inaccurate readings inimmunoassays for imatinib or its pharmacologically active salts.

DETAILED DESCRIPTION

In accordance with this invention, a new class of antibodies is providedwhich substantially selectively reacts with imatinib or itspharmacologically active salts and do not substantially react or crossreact with its metabolites as mentioned hereinabove. It has beendiscovered that through the use of these derivatives of imatinib offormula IV as immunogens, this new class of antibodies of this inventionis provided. It is through the use of these antibodies that animmunoassay, including reagents and kits for such immunoassay fordetecting and/or quantifying imatinib and its pharmacologically activesalts in blood, plasma or other body fluid samples has been developed.By use of this immunoassay, the presence and amount of imatinib or itspharmacologically active salts in body fluid samples of patients beingtreated this chemotherapeutic agent can be detected and/or quantified.In this manner, a patient being treated with imatinib or itspharmacologically active salts can be monitored during therapy and histreatment adjusted in accordance with said monitoring. By means of thisinvention one achieves the therapeutic drug management of imatinib orits pharmacologically active salts in cancer patients being treated withimatinib or its pharmacologically active salts as a chemotherapeuticagent.

The chemotherapeutic agent to be detected is imatinib of formula I orits pharmacologically active salts. These salts include the acidaddition salts for example salts for example with inorganic acids, suchas hydrochloric acid, sulfuric acid or a phosphoric acid, or withsuitable organic carboxylic or sulfonic acids, for example aliphaticmono- or di-carboxylic acids, such as trifluoroacetic acid, acetic acid,propionic acid, glycolic acid, succinic acid, maleic acid, fumaric acid,hydroxymaleic acid, malic acid, tartaric acid, citric acid or oxalicacid, or amino acids such as arginine or lysine, aromatic carboxylicacids, such as benzoic acid, 2-phenoxy-benzoic acid, 2-acetoxybenzoicacid, salicylic acid, 4-aminosalicylic acid, aromatic-aliphaticcarboxylic acids, such as mandelic acid or cinnamic acid, heteroaromaticcarboxylic acids, such as nicotinic acid or isonicotinic acid, aliphaticsulfonic acids, such as methane-, ethane- or 2-hydroxyethane-sulfonicacid, or aromatic sulfonic acids, for example benzene-, p-toluene- ornaphthalene-2 sulfonic acid. The preferred acid is methane sulfonicacid.

The reagents utilized in the assay of this invention are conjugates of acarrier having a reactive thiol or amino functional group with thecompounds of formula IV. These conjugates are competitive bindingpartners with the imatinib and its pharmacologically active saltspresent in the sample for the binding with the antibodies of thisinvention. Therefore, the amount of conjugate reagent which binds to theantibody will be inversely proportional to the amount of imatinib andits pharmacologically active salts in the sample. In accordance withthis invention, the assay utilizes any conventional measuring means fordetecting and measuring the amount of said conjugate which is bound orunbound to the antibody. Through the use of said means, the amount ofthe bound or unbound conjugate can be determined. Generally, the amountof imatinib or its pharmacologically active salts in a sample isdetermined by correlating the measured amount of the bound or unboundconjugate produced by the imatinib or its pharmacologically active saltsin the sample with values of the bound or unbound conjugate determinedfrom standard or calibration curve obtained with samples containingknown amounts of imatinib or its pharmacologically active salts, whichknown amounts are in the range expected for the sample to be tested.These studies for producing calibration curves are determined using thesame immunoassay procedure as used for the sample.

Definitions

Throughout this description the following definitions are to beunderstood:

The term “Ph” as used throughout this application designates a phenylradical. The term “alkylene” designates a divalent saturated straight orbranch chain hydrocarbon substituent containing from one to ten carbonatoms

The terms “immunogen” and “immunogenic” refer to substances capable ofeliciting, producing, or generating an immune response in an organism.

The term “conjugate” refers to any substance formed from the joiningtogether of separate parts. Representative conjugates in accordance withthe present invention include those formed by the joining together of asmall molecule, such as the compound of formula IV, and a largemolecule, such as a carrier carrier having one or more reactive thiol oramino functional group, which carrier can be a polyamine polymer,particularly protein. In the conjugate the small molecule maybe joinedat one or more active sites on the large molecule. The term conjugateincludes the term immunogen.

“Haptens” are partial or incomplete antigens. They are protein-freesubstances, mostly low molecular weight substances, which are notcapable of stimulating antibody formation, but which do react withantibodies. The latter are formed by coupling a hapten to a highmolecular weight immunogenic carrier and then injecting this coupledproduct, i.e., immunogen, into a human or animal subject. The hapten ofthis invention is imatinib or its pharmacologically active salts.

As used herein, a “spacing group” or “spacer” refers to a portion of achemical structure which connects two or more substructures such ashaptens, carriers, immunogens, labels, or tracers through a functionallinking group. These spacer groups will be enumerated hereinafter inthis application. The atoms of a spacing group and the atoms of a chainwithin the spacing group are themselves connected by chemical bonds.Among the preferred spacers are straight or branched, saturated orunsaturated, carbon chains. Theses carbon chains may also include one ormore heteroatoms within the chain or at termini of the chains. By“heteroatoms” is meant atoms other than carbon which are chosen from thegroup consisting of oxygen, nitrogen and sulfur. Spacing groups may alsoinclude cyclic or aromatic groups as part of the chain or as asubstitution on one of the atoms in the chain.

The number of atoms in the spacing group is determined by counting theatoms other than hydrogen. The number of atoms in a chain within aspacing group is determined by counting the number of atoms other thanhydrogen along the shortest route between the substructures beingconnected. A functional linking group may be used to activate, e.g.,provide an available functional site on, a hapten or spacing group forsynthesizing a conjugate of a hapten with a label or carrier orpolyamine polymer.

An “immunogenic carrier,” as the terms are used herein, is animmunogenic substance, commonly a protein or a protein modified tocontain a reactive thiol or amino group, that can join at one or morepositions with a hapten, in this case imatinib, thereby enabling thesehapten derivatives to induce an immune response and elicit theproduction of antibodies that can bind specifically with these haptens.The immunogenic carriers and the linking groups will be enumeratedhereinafter in this application. Among the immunogenic carriersubstances are included proteins, glycoproteins, complexpolyamino-polysaccharides, particles, and nucleic acids that arerecognized as foreign and thereby elicit an immunologic response fromthe host. The polyamino-polysaccharides may be prepared frompolysaccharides using any of the conventional means known for thispreparation.

Also various protein types may be employed as a poly(amino acid)immunogenic carrier. These types include albumins, serum proteins,lipoproteins, etc. Illustrative proteins include bovine serum albumin(BSA), keyhole limpet hemocyanin (KLH), egg ovalbumin, bovinethyroglobulin (BTG) etc. Alternatively, synthetic poly(amino acids) maybe utilized.

Immunogenic carriers can also include poly amino-polysaccharides, whichare a high molecular weight polymer built up by repeated condensationsof monosaccharides. Examples of polysaccharides are starches, glycogen,cellulose, carbohydrate gums such as gum arabic, agar, and so forth. Thepolysaccharide also contains polyamino acid residues and/or lipidresidues.

The immunogenic carrier can also be a poly(nucleic acid) either alone orconjugated to one of the above mentioned poly(amino acids) orpolysaccharides.

The immunogenic carrier can also include solid particles. The particlesare generally at least about 0.02 microns (μm) and not more than about100 μm, and usually about 0.05 μm to 10 μm in diameter. The particle canbe organic or inorganic, swellable or non-swellable, porous ornon-porous, optimally of a density approximating water, generally fromabout 0.7 to 1.5 g/mL, and composed of material that can be transparent,partially transparent, or opaque. The particles can be biologicalmaterials such as cells and microorganisms, including non-limitingexamples such as erythrocytes, leukocytes, lymphocytes, hybridomas,Streptococcus, Staphylococcus aureus, E. coli, and viruses. Theparticles can also be comprised of organic and inorganic polymers,liposomes, latex, phospholipid vesicles, or lipoproteins.

“Poly(amino acid)” or “polypeptide” is a polyamide formed from aminoacids. Poly(amino acids) will generally range from about 2,000 molecularweight, having no upper molecular weight limit, normally being less than10,000,000 and usually not more than about 600,000 daltons. There willusually be different ranges, depending on whether an immunogenic carrieror an enzyme is involved.

A “peptide” is any compound formed by the linkage of two or more aminoacids by amide (peptide) bonds, usually a polymer of α-amino acids inwhich the α-amino group of each amino acid residue (except the NH₂terminus) is linked to the α-carboxyl group of the next residue in alinear chain. The terms peptide, polypeptide and poly(amino acid) areused synonymously herein to refer to this class of compounds withoutrestriction as to size. The largest members of this class are referredto as proteins.

A “label,” “detector molecule,” or “tracer” is any molecule whichproduces, or can be induced to produce, a detectable signal. The labelcan be conjugated to an analyte, immunogen, antibody, or to anothermolecule such as a receptor or a molecule that can bind to a receptorsuch as a ligand, particularly a hapten. Non-limiting examples of labelsinclude radioactive isotopes, enzymes, enzyme fragments, enzymesubstrates, enzyme inhibitors, coenzymes, catalysts, fluorophores, dyes,chemiluminescers, luminescers, or sensitizers; a non-magnetic ormagnetic particle, a solid support, a liposome, a ligand, or a receptor.

The term “antibody” refers to a specific protein binding partner for anantigen and is any substance, or group of substances, which has aspecific binding affinity for an antigen to the exclusion of othersubstances. The generic term antibody subsumes polyclonal antibodies,monoclonal antibodies and antibody fragments.

The term “derivative” refers to a chemical compound or molecule madefrom a parent compound by one or more chemical reactions.

The term “carrier” refers to solid particles and/or polymeric polymershaving a reactive thiol or amino functional group such as immunogenicpolymers such as those mentioned above. Where the carrier is a solidparticle, the solid particle may be bound, coated with or otherwiseattached to a polyamine polymer to provide one or more reactive sitesfor bonding to the terminal functional group X in the compounds of theformula IV. On the other hand the immunoassay of this invention can becarried out by coating the antibody on the solid particles.

The term “reagent kit,” or “test kit,” refers to an assembly ofmaterials that are used in performing an assay. The reagents can beprovided in packaged combination in the same or in separate containers,depending on their cross-reactivities and stabilities, and in liquid orin lyophilized form. The amounts and proportions of reagents provided inthe kit can be selected so as to provide optimum results for aparticular application. A reagent kit embodying features of the presentinvention comprises antibodies specific for Imatinib or itspharmacologically active salts. The kit may further comprise ligands ofthe analyte and calibration and control materials. The reagents mayremain in liquid form or may be lyophilized.

The phrase “calibration and control materials” refers to any standard orreference material containing a known amount of a drug to be measured.The concentration of drug is calculated by comparing the resultsobtained for the unknown specimen with the results obtained for thestandard. This is commonly done by constructing a calibration curve.

The term “biological sample” includes, but is not limited to, anyquantity of a substance from a living thing or formerly living thing.Such living things include, but are not limited to, humans, mice,monkeys, rats, rabbits, horses, and other animals. Such substancesinclude, but are not limited to, blood, serum, plasma, urine, cells,organs, tissues, bone, bone marrow, lymph, lymph nodes, synovial tissue,chondrocytes, synovial macrophages, endothelial cells, and skin.

Reagents and Immunogens

In an immunoassay based upon an antibody, a conjugate of imatinib isconstructed to compete with the imatinib and its pharmacologicallyactive salts in the sample for binding sites on the antibody. In theimmunoassay of this invention, the reagents of formula IV are thenitrogen substituted imatinib derivatives formed on the amide bridge ofimatinib of formula I. In the compounds of formula IV, the linker spacerconstitutes the “Y-X” portion of this molecule. This linker X and thespacer—“Y” are conventional in preparing conjugates for immunoassays andimmunogens for producing antibodies. Any of the conventionalspacer-linking groups utilized to prepare conjugates for immunoassaysand immunogens for producing antibodies can be utilized in the compoundsof formula IV. Such conventional linkers and spacers are disclosed inU.S. Pat. No. 5,501,987 and U.S. Pat. No. 5,101,015.

The conjugates as well as the immunogens, are prepared from the compoundof the formula I. In the conjugates or immunogens of the carrier withthe hapten, the carriers are linked in one or positions to one or morereactive amino or thiol groups contained by the polymeric portion of thecarrier to the hapten which has the formula:

-   -   wherein X′ is a functional linking group capable of binding to        said carrier through said amino or thiol group and p and Y are        as above;

Among the preferred spacer groups are included the spacer groupshereinbefore mentioned. Particularly preferred spacing groups are groupssuch as alkylene containing from 1 to 10 carbon atoms,

wherein n and o are integers from 0 to 6, and m is an integer from 1 to6 with alkylene being the especially preferred spacing group.

In the compounds of formula IV-A, where X′ is a functional group linkingthe spacer, preferably through a reactive amine or thiol group on thepolymeric carrier. The group X′ is the result of the terminal functionalgroup X in the compounds of formula IV binding to the reactive amino orthiol group in the polymer of the carrier or the immunogen. Any terminalfunctional group capable of reacting with an amino or thiol group can beutilized as the functional group X in the compounds of formula IV. Theseterminal functional groups preferably included within X are:

wherein R₃ is hydrogen or taken together with its attached oxygen atomforms a reactive ester and R₄ is oxygen or sulfur. The radical—N═C═R_(4′) can be an isocyanate or as isothiocyanate. The active estersformed by OR₃ include imidoester, such as N-hydroxysuccinamide,1-hydroxy benzotriazole and p-nitrophenyl ester. However any activeester which can react with an amine group can be used.

When X in the compound of formula IV is

these compounds preferably react with the free amino group of thepolymeric or immunogenic carrier. On the other hand, when X in thecompound of formula IV is the maleimide radical of the formula

this compound preferably reacts with the thiol (or SH) group which maybe present on the polymeric or protein carrier, including theimmunogens, to produce the maleimide functional group as X′ in thecompounds of the formula IV-A.

In accordance with an embodiment, of this invention where X′ is amaleimide in the compounds of formula IV is attached to a polymericprotein which has been modified to convert a reactive amino group to athiol group. This can be done by the reacting a free amino group of apolymeric protein carrier with a compound of the formula

-   -   wherein R₁₅ is a thiol protecting group;    -   R₃ is as above; and    -   v is an integer of from 1 to 4.

In this manner, the thiol group, SH— becomes the functional group of thecarrier bonded to the remainder of the carrier. The reaction to convertthe reactive amino group of the protein is carried out in an aqueousmedium by mixing the protein containing carrier with the compound offormula VI in an aqueous medium. In this reaction temperature andpressure are not critical and the reaction can be carried out at roomtemperature and atmospheric pressure. Temperatures of from 10° C. to 25°C. are generally preferred. In the next step before the thiol modifiedcarrier is reacted with the compound of formula IV after the thiolprotecting group of carrier is removed by conventional means from theresulting reaction product of the compound of formula V with the proteincarrier.

Any conventional means for removing a thiol protecting group can beutilized in carrying out this reaction. However, in utilizing a means toremove the thiol protecting group, care must be taken that the reactantsbe soluble in the aqueous medium and do not in any way destroy or harmthe polyamine polymer contained in the carrier. A preferred means forremoving this protecting group is by the use of dithiothreitol as anagent to reduce the resultant condensation product. This reduction canbe carried out by simply adding the reducing agent to the reactionmedium without utilizing higher pressures or temperatures. Thisreduction can be carried out at room temperature and atmosphericpressure. Any conventional thiol protecting agent can be utilized in thecompound of formula VI. The thiol protecting groups are well known inthe art with 2-pyridyldithio being the preferred protecting group.

While the above method represents one means for converting a reactiveterminal amino group on the polyamine polymeric containing carrier to athiol group, any conventional means for carrying out this conversion canbe utilized. Methods for converting terminal amino groups on polyaminepolymeric containing carriers to thiols are well known in the art andcan be employed in accordance with this invention.

The reaction of the polymeric polyamine containing carrier having aterminal reactive thiol group with the compound of formula IV where X isa functional group capable of binding to the terminal thiol groupcarried by the carrier can be carried out by conventional means. In thepreferred embodiment the maleimide of carried by X in the compoundformula IV is reacted with the thiol group carried by the polyaminepolymeric carrier. Any well known means for addition of a thiol across amaleimide double bond can be utilized in producing the conjugates offormula VI A which are conjugated through a thiol bridge.

The carboxylic group and the active esters are coupled to the carrier orimmunogenic polymer by conventional means. The amine group on thepolyamine polymer, such as a protein, produces an amide group whichconnects the spacer to the polymer, immunogens or carrier and/orconjugates of this invention.

In the immunogens and conjugates of the present invention, the chemicalbonds between the carboxyl group-containing imatinib hapten and thereactive amino groups on the polyamine polymer contained by the carrieror immunogen can be established using a variety of methods known to oneskilled in the art. It is frequently preferable to form amide bonds.Amide bonds are formed by first activating the carboxylic acid moiety inthe compounds of formula IV-A by reacting the carboxy group with aleaving group reagent (e.g., N-hydroxysuccinimide,1-hydroxybenzotriazole, p-nitrophenol and the like). An activatingreagent such as dicyclohexylcarbodiimide, diisopropylcarbodiimide andthe like can be used. The activated form of the carboxyl group in theimatinib hapten of formula VI-A is then reacted with a buffered solutioncontaining the carrier with the reactive amino group.

On the other hand where X is a terminal isocyanate or thioisocyanateradical in the compound of formula IV, these radicals when reacted withthe free amine of a polyamine polymer produce the conjugate or immunogenof formula IV-A where X′ is

with the amino group on the polyamine carrier or the immunogenicpolypeptide.

Where X, in the compounds of formula IV contains an aldehyde radical,these compounds may be connected to the free amino group of thepolyamine polypeptide on the carrier through an amine linkage byreductive amination. Any conventional method of condensing an aldehydewith an amine such as through reductive amination can be used to formthis linkage. In this case, X′ in the ligand portions of formula IV is—CH₂—.

The compounds of formula IV are formed by reacting imatinib of formula Iwith a halide of the formula:halo-CH₂—(Y)_(p)—X  VII

-   -   wherein p, Y and X are as above

to form the compound of formula IV. Any conventional means of reacting ahalide with the nitrogen on the amide can be utilized in condensing thecompound of formula VII to this amide position on the imatinib offormula I. The use of a halide in the compound of formula VII providesan efficient means for forming such a substituted amide by condensingwith the amide group on the compound of formula I.

Where the compound of formula I is in the form of its salt it isnecessary to convert this salt to its free base before reacting with thecompound of formula V to form the compound of formula IV. This can becarried out by conventional means such as neutralization of the salt.Where the salt is a basic salt, neutralization can be accomplished in anaqueous media by addition of an acid. Where the salt is an acid additionsalt neutralization is accomplished in an aqueous media by addition of abase.

The compound of formula IV can be converted into the immunogens and/orthe conjugate reagents of this invention by reacting these compoundswith a carrier containing a polyamine or a polypeptide. The samepolypeptide can be utilized as the carrier and as the immunogenicpolymer in the immunogen of this invention provided that polyamine orpolypeptide is immunologically active. However, to form the conjugates,these polymers need not produce an immunological response as needed forthe immunogens. In accordance with this invention, the variousfunctional group represented by X in the compounds of formula IV can beconjugated to the carrier containing polymer with a reactive amino groupby conventional means of attaching a functional group to an amino groupcontained within the polymer. In accordance with a preferred embodiment,in the compound of formula IV where the attachment is through a reactiveamino group on the carrier, X is a carboxylic acid group or an activeester thereof.

Antibodies

The present invention also relates to novel antibodies includingmonoclonal antibodies to imatinib or its pharmacologically active saltsproduced by utilizing the aforementioned immunogens. In accordance withthis invention it has been found that these antibodies produced inaccordance with this invention are selectively reactive with imatinib orits pharmacologically active salts and do not react with N-desmethylimatinib which interferse with immunoassays for imatinib and itspharmacologically active salts. The ability of the antibodies of thisinvention not to react with N-desmethyl imatinib makes these antibodiesprovide an immunoassay for detecting the presence and/or quantifying theamount of imatinib and its pharmacologically active salts in patientfluid samples

The present invention relates to novel antibodies and monoclonalantibodies to imatinib or its pharmacologically active salts. Theantisera of the invention can be conveniently produced by immunizinghost animals with the immunogens of this invention. Suitable hostanimals include rodents, such as, for example, mice, rats, rabbits,guinea pigs and the like, or higher mammals such as goats, sheep, horsesand the like. Initial doses, bleedings and booster shots can be givenaccording to accepted protocols for eliciting immune responses inanimals, e.g., in a preferred embodiment mice received an initial doseof 100 ug immunogen/mouse, i.p. and two or more subsequent booster shotsof between 50 and 100 ug immunogen/mouse over a six month period.Through periodic bleeding, the blood samples of the immunized mice wereobserved to develop an immune response against imatinib or itspharmacologically active salts binding utilizing conventionalimmunoassays. These methods provide a convenient way to screen for hostswhich are producing antisera having the desired activity. The antibodieswere also screened against the major metabolites of imatinib or itspharmacologically active salts and showed no substantial binding tothese compounds.

Monoclonal antibodies are produced conveniently by immunizing Balb/cmice according to the above schedule followed by injecting the mice with100 ug immunogen i.p. or i.v. on three successive days starting fourdays prior to the cell fusion. Other protocols well known in theantibody art may of course be utilized as well. The completeimmunization protocol detailed herein provided an optimum protocol forserum antibody response for the antibody to imatinib or itspharmacologically active salts.

B lymphocytes obtained from the spleen, peripheral blood, lymph nodes orother tissue of the host may be used as the monoclonal antibodyproducing cell. Most preferred are B lymphocytes obtained from thespleen. Hybridomas capable of generating the desired monoclonalantibodies of the invention are obtained by fusing such B lymphocyteswith an immortal cell line, which is a cell line that which imparts longterm tissue culture stability on the hybrid cell. In the preferredembodiment of the invention the immortal cell may be a lymphoblastoidcell or a plasmacytoma cell such as a myeloma cell, itself an antibodyproducing cell but also malignant. Murine hybridomas which produceImatinib or its pharmacologically active salts monoclonal antibodies areformed by the fusion of mouse myeloma cells and spleen cells from miceimmunized against Imatinib or its pharmacologically active salts-proteinconjugates. Chimeric and humanized monoclonal antibodies can be producedby cloning the antibody expressing genes from the hybridoma cells andemploying recombinant DNA methods now well known in the art to eitherjoin the subsequence of the mouse variable region to human constantregions or to combine human framework regions with complementarydetermining regions (CDR's) from a donor mouse or rat immunoglobulin. Animproved method for carrying out humanization of murine monoclonalantibodies which provides antibodies of enhanced affinities is set forthin International Patent Application WO 92/11018.

Polypeptide fragments comprising only a portion of the primary antibodystructure may be produced, which fragments possess one or moreimmunoglobulin activities. These polypeptide fragments may be producedby proteolytic cleavage of intact antibodies by methods well known inthe art, or by inserting stop codons at the desired locations inexpression vectors containing the antibody genes using site-directedmutageneses to produce Fab fragments or (Fab′)₂ fragments. Single chainantibodies may be produced by joining VL and VH regions with a DNAlinker (see Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85:5879-5883(1988) and Bird et al., Science, 242:423-426 (1988))

The antibodies of this invention are selective for imatinib and itspharmacologically active salts without having any substantialcross-reactivity with N-des methyl imatinib. In addition theseantibodies display substantially no cross reactivity with any of itstherapeutically active or inactive imatinib metabolites. By having nosubstantial cross-reactivity it is meant that the antibodies of thisinvention have a cross reactivity relative to imatinib and itspharmacologically active salts with these metabolites, particularlyN-des methyl imatinib, of less than 15%, preferably less than 10%.

Immunoassays

In accordance with this invention, the conjugates and the antibodiesgenerated from the immunogens of these compounds of formula IV can beutilized as reagents for the determination of imatinib or itspharmacologically active salts in patient samples. This determination isperformed by means of an immunoassay. Any immunoassay in which thereagent conjugates formed from the compounds of formula IV compete withthe imatinib or its pharmacologically active salts in the sample forbinding sites on the antibodies generated in accordance with thisinvention can be utilized to determine the presence of imatinib or itspharmacologically active salts in a patient sample. The manner forconducting such an assay for imatinib or its pharmacologically activesalts in a sample suspected of containing imatinib or itspharmacologically active salts, comprises combining an (a) aqueousmedium sample, (b) an antibody to imatinib or its pharmacologicallyactive salts generated in accordance with this invention and (c) theconjugates formed from the compounds of formula IV or mixtures thereof.The amount of imatinib or its pharmacologically active salts in thesample can be determined by measuring the inhibition of the binding tothe specific antibody of a known amount of the conjugate added to themixture of the sample and antibody. The result of the inhibition of suchbinding of the known amount of conjugates by the unknown sample iscompared to the results obtained in the same assay by utilizing knownstandard solutions of imatinib or its pharmacologically active salts. Indetermining the amount of imatinib or its pharmacologically active saltsin an unknown sample, the sample, the conjugates formed from thecompounds of formula IV and the antibody may be added in any order.

Various means can be utilized to measure the amount of conjugate formedfrom the compounds of formula IV bound to the antibody. One method iswhere binding of the conjugates to the antibody causes a decrease in therate of rotation of a fluorophore conjugate. The amount of decrease inthe rate of rotation of a fluorophore conjugate in the liquid mixturecan be detected by the fluorescent polarization technique such asdisclosed in U.S. Pat. No. 4,269,511 and U.S. Pat. No. 4,420,568.

On the other hand, the antibody can be coated or absorbed onnanoparticles so that when these particles react with the imatinib orits pharmacologically active salts conjugates formed from the compoundsof formula IV, these nanoparticles form an aggregate. However, when theantibody coated or absorbed nanoparticles react with the imatinib or itspharmacologically active salts in the sample, the imatinib or itspharmacologically active salts from the sample bound to thesenanoparticles does not cause aggregation of the antibody nanoparticles.The amount of aggregation or agglutination can be measured in the assaymixture by absorbance.

On the other hand, these assays can be carried out by having either theantibody or the imatinib or its pharmacologically active saltsconjugates attached to a solid support such as a microtiter plate or anyother conventional solid support including solid particles. Attachingantibodies and proteins to such solid particles is well known in theart. Any conventional method can be utilized for carrying out suchattachments. In many cases, in order to aid measurement, labels may beplaced upon the antibodies, conjugates or solid particles, such asradioactive labels or enzyme labels, as aids in detecting the amount ofthe conjugates formed from the compounds of formula IV which is bound orunbound with the antibody. Other suitable labels include chromophores,fluorophores, etc.

As a matter of convenience, assay components of the present inventioncan be provided in a kit, a packaged combination with predeterminedamounts of new reagents employed in assaying for imatinib or itspharmacologically active salts. These reagents include the antibody ofthis invention, as well as, the conjugates formed from the compounds offormula IV.

In addition to these necessary reagents, additives such as ancillaryreagents may be included, for example, stabilizers, buffers and thelike. The relative amounts of the various reagents may vary widely toprovide for concentrations in solution of the reagents whichsubstantially optimize the sensitivity of the assay. Reagents can beprovided in solution or as a dry powder, usually lyophilized, includingexcipients which on dissolution will provide for a reagent solutionhaving the appropriate concentrations for performing the assay.

Examples

In the examples, the following abbreviations are used for designatingthe following:

-   -   NaH sodium hydride    -   THF tetrahydrofuran    -   DMF dimethylformamide    -   LiOH lithium hydroxide    -   EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride    -   Sulfo-NHS N-hydroxysulfosuccinimide    -   DMSO dimethylsulfoxide    -   MeOH methanol    -   CH₂Cl₂ dichloromethane    -   APCI atmospheric pressure chemical ionizatin mass spectrometry    -   TLC thin layer chromatography    -   HOAc acetic acid    -   CHCl₃ chloroform    -   HPLC high pressure liquid chromatography    -   ANS 8-Anilino-1-naphthalenesulfonic acid    -   HRP horse radish-peroxidase    -   TMB 3,3′,5,5′-Tetramethylbenzidine    -   TRIS Tris(hydroxymethyl)aminomethane hydrochloride    -   BSA bovine serum albumin    -   KLH Keyhole Limpet Hemocyanin    -   PBS phosphate buffered saline    -   diH₂O deionized water

In the examples, Scheme 1 below set forth the specific compoundsprepared and referred to by numbers in the Examples. The phosphatebuffer composition has an aqueous solution containing

15.4 mM Sodium phosphate dibasic (Na₂HPO₄)

4.6 mM Sodium phosphate monobasic (NaH₂PO₄)

pH=7.2±0.10

The ether used in the Examples was diethyl ether. Parts or percentagesgiven in these examples are parts by volume.

EXAMPLES Example 1 Extraction of Imatinib Mesylate

Fourteen tablets, each containing 400 mg of the imatinib as the mesylatesalt, were crushed to a fine powder with a mortar and pestle; the tabletcoating was not first removed. The powder of the crushed tablets wasstirred in 1500 mL 10% MeOH (by volume)/CH₂Cl₂ for four hours. Themixture from the crushed tablets containing imatinib mesylate wasfiltered through Celite and the solvent stripped off to yield 7.14 g ofa yellow solid containing imatinib mesylate. The yellow solid containingimatinibin mesylate was dissolved in 75 mL of warm 20% chloroform (byvolume) in ethanol to produce a solution of imatinib mesylate, then 50mL of 1:1 ether:ethanol (volume:volume) was added, causing the solutionof imatinib mesylate to become cloudy. Cooling in ice inducedprecipitation of the imatinib mesylate, which continued to progress withthe slow addition of ether. The precipatated imatinib mesylate waslayered with ether, covered, and allowed to stand overnight.

The precipatated imatinib mesylate was collected by filtration andwashed with 50 mL cold 10% (by volume) ethanol in ether, then driedunder vacuum to yield 6.42 g (115%) of imatinib mesylate as a lightyellow solid. Recrystallization of imatinib mesylate fromchloroform/ethanol with the aid of ether was carried out a second timeto yield 4.45 g (79%) of imatinib mesylate as a light yellow solid. Thislight yellow solid of imatinib mesylate isolated from the tablets wasused in Example 2. The structure was confirmed by NMR and elementalanalysis. The purity was confirmed by HPLC.

Example 2 Preparation of Imatinib Free Base

The imatinib mesylate salt (1) (1.01 g, 1.71 mmol) prepared in Example 1was added to 250 mL of dichloromethane to form a suspension of imatinibmesylate. 50 mL of 10% saturated aqueous NaHCO₃ was added and mixed wellwith the suspension of imatinib mesylate in dichloromethane to producethe free base of imatinib in the organic layer (dichloromethane). Theemulsion formed from the aqueous NaHCO₃ and the dichloromethoane wasremoved by filtration, producing an organic layer of dichloromethanecontaining the imatinib as the free base and an aqueous layer. Theorganic layer of dicholomethane containing imatninb as the free base wasseparated from the aqueous layer. The organic layer was dried overNa₂SO₄/MgSO₄. To isolate the imatinib free base, the organic layer(dichloromethane) was filtered to remove the Na₂SO₄/MgSO₄ and thenstipped off, producing a solid containing the free base of imatinib.Toluene was added to the solid containing imatinib free base and flashevaporated three times and then dried under vacuum to remove anyresidual water. The free base of imatinib was obtained as a white solidand used in example 3. The free base of imatinib exhibited ¹H, ¹³C NMRand APCI data consistent with the structure. NMR assignments were basedon a DQF-COSY experiment.

Example 3 Preparation of Butyric Acid Ethyl Ester of Imatinib

The dried free base of imatinib prepared in example 2 was dissolved in35 mL dry DMF under nitrogen and cooled in ice. The mixture containingimatinib was stirred effectively and solid sodium hydride (60%dispersion in mineral oil, 0.111 g, 2.78 mmol, 1.6 eq) was added all atonce. A solution of ethyl 4-bromobutyrate (0.59 g, 3.0 mmol, 1.8 eq) in3.5 mL DMF was slowly added via syringe to the mixture containingimatinib, and the reaction to produce the butyric acid ethyl ester ofimatinib (2) was allowed to proceed overnight, warming to ambienttemperature with the bath. In situ analysis of the reaction mixture byAPCI(+) showed m/z=608.3 (100%), 494.2 (30%), and 722.4 (5%) amu,corresponding to the butyric acid ethyl ester of imatinib (2), imatinibstarting material, and a di-butyric acid ethyl ester of imatinib,respectively.

The reaction mixture was diluted with 100 mL dichloromethane, cooled inice and quenched with 10 mL water. The dichloromethane containing thebutric acid ethyl esters of imatinib and aqueous layers were separated.To increase the yield the aqueous layer was extracted with 3×50 mLdichloromethane and 2×25 mL ethyl acetate. The combined organicfractions containing the butyric acid ethyl ester derivates of imatinibwere stripped leaving a mixture of imatinib derivatives. To isolate thebutyric acid ethyl ester of imatinib, the mixture was chromatographed on100 g silica gel using a 5-50% methanol/dichloromethane gradient. Theresulting butyric acid ethyl ester of imatinib (2) had mass 0.61 g (59%)TLC R_(f)=0.36 (1:4:0.05 MeOH:CHCl₃:HOAc). APCI (+) m/z=608.3 amu.

Example 4 Hydrolysis of Imatinib Butyric Acid Ethyl Ester

4-{[4-(4-methyl-piperazin-1-ylmethyl)-benzoyl]-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-amino}-butyricacid ethyl ester (2) (1.21 g, 2.0 mmol, 1 eq) was dissolved in 60 mL THFand cooled in ice. A solution of LiOH.H₂O (0.28 g, 6.7 mmol, 3.4 eq) in20 mL water was added dropwise while stirring. This hydrolysis reactionwas incubated for 3 h at 0° C. and then allowed to proceed overnight,while warming to room temperature to produce the free acid, imatinbbutyric acid.

After cooling the reaction mixture containing the imatinb butyric acidin ice, 0.5 M HCl (aq) was added dropwise to obtain a pH 5-6. Thevolatile solvents were stripped to afford a crude reaction productcontaining the imatinib butyric acid as a white solid. To removeimpurities the crude reaction product was triturated in water, collectedby filtration, and dried under vacuum and then triturated in ether toprovide the butyric acid derivative of imatinib, compound 3, as a yellowsolid, mass 0.99 g (86%) as the partial HCl salt. Melting point:203-207° C. (dec). TLC R_(f)=0.26 (7:1:0.1) CH₂Cl₂:MeOH:NH₄OH. APCI (+)m/z=580.2 amu. ¹H NMR (dmso-d₆, 80° C.) is consistent with thestructure. Analysis for C₃₃H₃₇N₇O₃.1.65H₂O.0.1HCl requires C, 64.48; H,6.25; N, 15.83; Cl, 0.63. Found C, 64.65; H, 6.64; N, 15.99; Cl, 0.58.HPLC purity was 100%.

Example 5 Preparation of Sulfo-NHS Activated Ester of Imatinib

Imatinib butyric acid (3) was derivatized by reaction with EDC andsulfo-NHS to produce the sulfo-NHS activated ester of imatinib foreventual conjugation to the proteins (examples 6a, b, 7). To a stirred 5mL of anhydrous DMSO compound 3 (94 mg, 0.16 mmol) was added, followedby EDC (94 mg, 0.49 mmol) and sulfo-NHS (107 mg, 0.49 mmol). Thereaction mixture was stirred at room temperature for 18 hours undernitrogen to produce the sulfo-NHS activated ester of imatinib. Thereaction mixture was used directly in Examples 6a and 6b.

Example 6a Preparation of Imatinib Immunogen

A protein solution of KLH was prepared by dissolving 300 mg of KLH in19.6 mL of phosphate buffer (50 mM, pH 7.5) and then slowly adding 39.2mL of DMSO while stirring the protein solution of KLH on ice. Stirringwas continued for an additional 30 minutes at room temperature, followedby addition of sulfo-NHS activated imatinib derivative (4) prepared inExample 5 (2.532 mL, 0.08 mmol). The reaction mixture of KLH andactivated imatinib derivative (4) was allowed to stir for 18 hours atroom temperature in an amber glass bottle producing an imatinib-KLHconjugate (5). The imatinib-KLH conjugate was then purified by dialysisagainst 66% DMSO in phosphate buffer (50 mM, pH 7.5) at roomtemperature. Thereafter the DMSO proportion was reduced stepwise: 60%,50%, 40%, 20%, 10% and 0%. The last dialysis was performed againstphosphate buffer at 4° C. The imatinib-KLH conjugate (5) wascharacterized by ultraviolet-visible spectroscopy. The conjugate wasdiluted to a final concentration of 2 mg/mL in phosphate buffer (50 mM,pH 7.5).

Example 6b Preparation of Imatinib Immunogen

Imatinib immunogen was prepared as in Example 6a except the imatinib-KLHconjugate (5) was diluted to a final concentration of 2 mg/mL inphosphate buffer and DMSO (50% by volume).

Example 7 Preparation of Imatinib-BSA Conjugate with Derivative 4

A protein solution of BSA was prepared by dissolving 1,000 mg BSA inphosphate buffer (50 mM, pH 7.5) for a final concentration of 50 mg/mL.40 mL of DMSO was slowly added to the protein solution of BSA whilestirring on ice. Stirring was continued for an additional 30 minutes atroom temperature, followed by addition of sulfo-NHS activated imatinibderivative (4) prepared in Example 5 (0.437 mL, 0.014 mmol). The amountof sulfo-NHS activated imatinib derivative (4) added to the proteinsolution of BSA was calculated for a 1:1 molar ratio between thederivative of imatinib (4) and BSA. The mixture of BSA and activatedimatinib derivative (4) was allowed to stir for 18 hours at roomtemperature in an amber glass bottle to produce the conjugate of theactivated imatinib ester (4) and BSA. This conjugate was then purifiedby dialysis against 66% DMSO in phosphate buffer (50 mM, pH 7.5) at roomtemperature. Thereafter the DMSO proportion was reduced stepwise: 60%,50%, 40%, 20%, 10% and 0%. The last dialysis was performed againstphosphate buffer at 4° C. The purified imatinib-BSA conjugate wascharacterized by UV/VIS spectroscopy.

Example 8a Preparation of Polyclonal Antibodies to Imatinib

Two groups of ten female BALB/c mice were immunized i.p. one group with100 μg/mouse of imatinib-KLH immunogen as prepared in Example 6a (10mice) and the other group with 100 μg/mouse of imatinib-KLH immunogen(10 mice) emulsified in Complete Freund's adjuvant as prepared Example6b. The mice were boosted once four weeks after the initial injectionwith 100 μg/mouse of the same immunogens emulsified in IncompleteFreund's Adjuvant. Twenty days after the boost test bleeds containingpolyclonal antibodies from each mouse were obtained by orbital bleed.The anti-serum from these test bleeds contained imatinib antibodies wereevaluated in Examples 9, 10a and 11.

Example 8b Preparation of Monoclonal Antibodies to Imatinib

Mice from example 8a that were immunized with imatinib-KLH prepared in6b were used to produce monoclonal antibodies. For monoclonal antibodiesstarting three days before the fusion, the mice were injected i.p. with400 μg (3 days before fusion), 200 μg (2 days before fusion), and 200 μg(1 day before fusion) of imatinib-KLH in PBS/DMSO prepared in example6b. Spleen cells were isolated from the selected mice and fused with2×10⁷ SP2/0 cells with 50% polyethylene glycol 1500 according to themethod of Coligan, J. E. et al., eds., Current Protocols in Immunology,2.5.1-2.5.8, (1992), Wiley & Sons, NY. The fused cells were plated onten 96-well plates in DMEM/F12 supplemented with 20% FetalClone I, 2%L-glutamine (100 mM) and 2% 50× HAT. Two to three weeks later, thehybridoma supernatant was assayed for the presence of anti-imatinibantibodies by ELISA (as in example 10b). Cells from the wells that gavepositive ELISA results (example 10b) were expanded to 24 well plates.Clones positive by ELISA were subcloned twice by limiting dilutionaccording to the method disclosed in Coligan, J. E. et al., eds.,Current Protocols in Immunology, 2.5.8-2.5.17, (1992), Wiley & Sons, NY.Hybridoma culture supernatants containing monoclonal antibody fromselected subclones were confirmed for imatinib binding by a competitiveELISA (example 11). These monoclonal antibodies were tested for imatinibbinding and cross-reactivity to a major imatinib metabolite, N-desmethylimatinib, by indirect competitive microtiter plate assay as described inexample 11.

Example 9 Microtiter Plate Sensitization Procedure with Imatinib-BSAConjugate

The ELISA method for measuring imatinib concentrations was performed inpolystyrene microtiter plates (Nunc MaxiSorp F8 Immunomodules) optimizedfor protein binding and containing 96 wells per plate. Each well wascoated with imatinib-BSA conjugate (prepared as in Example 7) by adding300 μL of imatinib-BSA conjugate at 10 μg/mL in 0.05M sodium carbonate,pH 9.6, and incubating for three hours at room temperature. The wellswere washed with 0.05M sodium carbonate, pH 9.6 and then were blockedwith 375 μL of 5% sucrose, 0.2% sodium caseinate solution for 30 minutesat room temperature. After removal of the post-coat solution the plateswere dried at 37° C. overnight.

Example 10a Antibody Screening Procedure Titer

This procedure is to find the dilution of antibody to be tested fordisplacement as in Example 11. The ELISA method for screening imatinibantibodies (produced in Example 8a and 8b) was performed with themicrotiter plates that were sensitized with imatinib-BSA conjugateprepared in Example 9. The antibody screening assay was performed bydiluting the murine serum from test bleeds (as in Example 8a) containingpolyclonal imatinib antibodies to 1:2,000, 1:6,000, 1:20,000 and1:50,000 (volume/volume) in phosphate buffered saline containing 0.1%BSA and 0.01% thimerosal. For evaluation of monoclonal antibodies,hybridoma supernatants of Example 8b, which were found to be positivefor the presence of antibody by the procedure of Example 10b werediluted 1:2, 1:4, 1:16, etc. (volume/volume) in phosphate bufferedsaline containing 0.1% BSA and 0.01% thimerosal. To each well ofimatinib-BSA sensitized wells (prepared in example 9) 50 μL phosphatebuffered saline containing 0.1% BSA and 0.01% thimerosal and 50 μL ofdiluted antibody were added and incubated for 10 minutes at roomtemperature with shaking. During this incubation antibody binds to theimatinib-conjugate passively absorbed in the wells (Example 9). Thewells of the plates were washed three times with 0.02 M TRIS, 0.9% NaCl,0.5% Tween-80 and 0.001% thimerosal, pH 7.8 to remove any unboundantibody. To detect the amount of imatinib antibody bound to theimatinib-BSA conjugate in the wells, 100 μL of a goat anti-mouseantibody-HRP enzyme conjugate (Jackson Immunoresearch) diluted to aspecific activity (approximately 1/3000) in PBS with 0.1% BSA, 0.05%ANS, 0.01% thimerosal, capable of binding specifically with murineimmunoglobulins and producing a colored product when incubated with asubstrate, in this example TMB were added to each well. After anincubation of 10 minutes at room temperature with shaking, during whichthe goat anti-mouse antibody-HRP enzyme conjugate binds to imatinibantibodies in the wells, the plates were again washed three times toremove unbound goat anti-mouse antibody-HRP enzyme conjugate. To developa measurable color in the wells washing was followed by the addition of100 μL of TMB (TMB Substrate, BioFx), the substrate for HRP, to developcolor during a 10 minute incubation with shaking at room temperature.Following the incubation for color development, 50 μL of stop solution(1.5% sodium fluoride in di H₂O) was added to each well to stop thecolor development and after 20 seconds of shaking the absorbance wasdetermined at 650 nm (Molecular Devices Plate Reader). The amount ofantibody in a well was proportional to the absorbance measured and wasexpressed as the dilution (titer) resulting in an absorbance of 1.5.Titers were determined by graphing antibody dilution of the antibodymeasured (x-axis) vs. absorbance 650 nm (y-axis) and interpolating thetiter at an absorbance of 1.5. The titer which produced absorbance of1.5 determined the concentration (dilution) of antibody used in theindirect competitive microtiter plate assay described in Example 11.

Example 10b Antibody Screening Procedure Monoclonal Screening

The ELISA method for screening imatinib monoclonal antibodies (producedin example 8b) was performed with the microtiter plates that weresensitized with imatinib-BSA as described in example 9. To each well ofimatinib-BSA sensitized wells (prepared in example 9) 50 μL phosphatebuffered saline containing 0.1% BSA and 0.01% thimerosal and then 50 μLof monoclonal culture supernatant were added and incubated for 10minutes at room temperature with shaking. During this incubationantibody binds to the imatinib-conjugate in the well. The wells of theplates were washed three times with 0.02 M TRIS, 0.9% NaCl, 0.5%Tween-80 and 0.001% thimerosal, pH 7.8 to remove any unbound antibody.To detect the amount of imatinib antibody bound to the imatinib-BSAconjugate in the wells, 100 μL of a goat anti-mouse antibody-HRP enzymeconjugate (Jackson Immunoresearch) diluted 1/3000 in PBS with 0.1% BSA,0.05% ANS, 0.01% thimerosal, capable of binding specifically with murineimmunoglobulins and producing a colored product when incubated with asubstrate, in this example TMB, were added to each well. After anincubation of 10 minutes at room temperature with shaking, during whichthe goat anti-mouse antibody-HRP enzyme conjugate binds to imatinibantibodies in the wells, the plates were again washed three times toremove unbound goat anti-mouse antibody-HRP enzyme conjugate. To developa measurable color in the wells washing was followed by the addition of100 μL of TMB (TMB Substrate, BioFx), the substrate for HRP, to developcolor during a 10 minute incubation with shaking at room temperature.Following the incubation for color development, 50 μL of stop solution(1.5% sodium fluoride in di H₂O) was added to each well to stop thecolor development and after 10 seconds of shaking the absorbance wasdetermined at 650 nm (Molecular Devices Plate Reader). The amount ofantibody in a well was proportional to the absorbance measured. Sampleswith an absorbance of greater than three or more times background weredesignated as positive. Fifty samples with highest absorbance wereexpanded to 24 well plates, as described in Example 8b.

Example 11 Indirect Competitive Microtiter Plate Immunoassay ProcedureDetermining IC₅₀ and Cross-Reactivity for Antibodies to Imatinib

The ELISA method for determining IC₅₀ values and cross-reactivity wasperformed with the microtiter plates that were sensitized withimatinib-BSA described in Examples 9. Analytes—imatinib and N-desmethylimatinib were diluted in diH₂O over a concentration range of 10 to1,000,000 ng/mL. Each of the assays were performed by incubating 50 μLof the imatinib solution with 50 μL of one of the antibodies selectedfrom the polyclonal antibodies produced in Example 8a with the immunogenof Example 6a and those produced in Example 8a with the immunogen ofExample 6b and the monoclonal antibody produced in Example 8b. Theassays were all performed by diluting the concentration of theantibodies in each of the wells to the titer determined in Example 10a.During the 10 minute incubation (R.T., with shaking) there is acompetition of antibody binding for the imatinib-BSA conjugate in thewell (produced in example 9) and the analyte in solution. Following thisincubation the wells of the plate were washed three times with 0.02 MTRIS, 0.9% NaCl, 0.5% Tween-80 and 0.001% thimerosal, pH 7.8 to removeany material that was not bound. To detect the amount of imatinibantibody bound to the imatinib-BSA conjugate in the wells (produced inexample 9), 100 μL of a goat anti-mouse antibody-HRP enzyme conjugate(Jackson Immunoresearch) diluted to a predetermined specific activity(approximately 1/3000) in PBS with 0.1% BSA, 0.05% ANS, 0.01%thimerosal, capable of binding specifically with murine immunoglobulinsand producing a colored product when incubated with a substrate, in thisexample TMB, were added to each well. After an incubation of 10 minutesat room temperature with shaking, during which the goat anti-mouseantibody-HRP enzyme conjugate binds to imatinib antibodies in the wells,the plates were again washed three times to remove unbound secondaryconjugate. To develop a measurable color in the wells washing wasfollowed by the addition of 100 μL of TMB (TMB Substrate, BioFx), thesubstrate for HRP, to develop color in a 10 minute incubation withshaking at room temperature. Following the incubation for colordevelopment, 50 μL of stop solution (1.5% sodium fluoride in di H₂O) wasadded to each well to stop the color development and after 20 seconds ofshaking the absorbance was determined at 650 nm (Molecular Devices PlateReader). The amount of antibody in a well was proportional to theabsorbance measured and inversely proportional to the amount of imatinibin the sample. The IC₅₀'s of imatinib and N-desmethyl imatinib weredetermined by constructing dose-response curves with the absorbance inthe wells plotted versus analyte concentration in the wells. Theabsorbance of the color in the wells containing analyte was compared tothat with no analyte and a standard curve was generated. The IC₅₀ valuefor a given analyte was defined as the concentration of analyte that wasrequired to have 50% of the absorbance of the wells containing noanalyte. The cross-reactivity was calculated as the ratio of the IC₅₀for imatinib mesylate to the IC₅₀ for N-desmethyl imatinib and expressedas a percent. When measured with this pool of antibodies, the percentcross-reactivities relative to imatinib for N-desmethyl imatinib wereless than or equal to 7%. Results for polyclonal antibodies to imatinibare in table I below. When measured with selected monoclonal antibodiesthe percent cross-reactivities relative to imatinib for N-desmethylimatinib were less than 4%. Results for monoclonal antibodies toimatinib are in table II.

TABLE I Cross-reactivity of competitive immunoassay using polyclonalantibodies to imatinib (Example 8a). Bleed # 1 2 3 4 5 Produced from oneof two 6b 6b 6b 6a 6a groups of ten mice with immunogen prepared inexample: Analyte Imatinib 100% 100% 100% 100% 100% N-desmethyl imatinib 1%  5%  2%  7%  1%

TABLE II Cross-reactivity of competitive immunoassay using monoclonalantibodies to imatinib (Example 8b). Monoclonal antibody number Analyte9F2-4-29 9F2-4-31 Imatinib 100% 100% N-desmethyl imatinib  2.7%  3.2%

As seen from Tables I and II, the antibodies produced in accordance withthis invention which were substantially reactive with imatinib mesylateand substantially non-reactive with the metabolite, N-desmethylimatinib.

1. An immunoassay for detecting imatinib and its pharmacologicallyactive salts in a sample comprising providing a mixture of a sample, anantibody selectively reactive with imatinib and its pharmacologicallyactive salts and a conjugate of a carrier containing a polymer havingeither a reactive thiol or amino group with a compound of the formula:

or its pharmacologically active salts wherein Y is an organic spacinggroup; X is a terminal functional group capable of binding to saidcarrier through said amino or thiol group and; p is an integer from 0 to1; causing the imatinib and its pharmacologically active salts in thesample and said conjugate to bind with said antibody and thereaftermeasuring the amount of said conjugate in said mixture which is bound orunbound to said antibody whereby the presence of imatinib or itspharmacologically active salts in the sample can be determined.
 2. Theprocess of claim 1, wherein the sample is a human sample.
 3. Theimmunoassay of claim 2, wherein said antibody is generated from animmunogen comprising an immunogenic carrier containing a polymer havingeither a reactive thiol or amino group linked to a compound of theformula:

or its pharmacologically active salts. wherein Y is an organic spacinggroup; X is a terminal functional group capable of binding to saidcarrier through said amino or thiol group and; p is an integer from 0to
 1. 4. The immunoassay of claim 2, wherein the antibody or theconjugate is attached to a solid support.
 5. The immunoassay of claim 4,wherein the solid support is microtiter plates.
 6. The immunoassay ofclaim 2, wherein the solid support is nanoparticles.
 7. The immunoassayof claim 1 wherein said antibody does not substantially bind toN-desmethyl imatinib.